DNA Ssb Research Articles

CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences

The B DNA of the genome contains numerous sequences that can form various noncanonical structures including G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating-motif (i-motif, iM) formed by alternately arranged C-C+ pairs. One of the easy yet sensitive methods to study G4s and iMs is circular dichroism (CD) spectroscopy which generates characteristic G4 and iM peaks. We have analyzed and compared the effects of various environmental factors including pH, buffer composition, temperature, flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB) on the CD patterns of G4s and iMs generated by two groups of DNA molecules, one containing tandem repeats of GGGGCC and CCCCGG from the C9ORF72 gene associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and the second containing polyG/polyC clusters from oncogene promoter-proximal regions without such tandem repeats. Changes in pH caused drastic changes in CCCCGG-iM and GGGGCC-G4 and the changes were dependent on repeat numbers and G:C base-pairing. In contrast, with the DNA sequences from the promoter-proximal regions of oncogenes, iMs disassembled upon pH changes with the peak slowly shifting to lower wavelength but the G4s did not show significant change. Complementary DNA strands and flanking DNA sequences also regulate G4 and iM formation. The single-stranded DNA binding protein, SSB, disassembled both G4s and iMs formed by almost all sequences suggesting an in vivo role for SSBs in the disassembly of G4s and iMs during DNA replication and other DNA transactions.

Arid1a Loss Enhances Disease Progression in a Murine Model of Osteosarcoma.

Osteosarcoma is an aggressive bone malignancy, molecularly characterized by acquired genome complexity and frequent loss of TP53 and RB1. Obtaining a molecular understanding of the initiating mutations of osteosarcomagenesis has been challenged by the difficulty of parsing between passenger and driver mutations in genes. Here, a forward genetic screen in a genetic mouse model of osteosarcomagenesis initiated by Trp53 and Rb1 conditional loss in pre-osteoblasts identified that Arid1a loss contributes to OS progression. Arid1a is a member of the canonical BAF (SWI/SNF) complex and a known tumor suppressor gene in other cancers. We hypothesized that the loss of Arid1a increases the rate of tumor progression and metastasis. Phenotypic evaluation upon in vitro and in vivo deletion of Arid1a validated this hypothesis. Gene expression and pathway analysis revealed a correlation between Arid1a loss and genomic instability, and the subsequent dysregulation of genes involved in DNA DSB or SSB repair pathways. The most significant of these transcriptional changes was a concomitant decrease in DCLRE1C. Our findings suggest that Arid1a plays a role in genomic instability in aggressive osteosarcoma and a better understanding of this correlation can help with clinical prognoses and personalized patient care.

A Molecular Approach to Characterize the Effects of Fluence, Fluence Rate and LET on Radiation Damage

Effects of fluence, fluence rate and LET on radiation damage are not resolved using traditional methods of measuring energy deposited by ionization events. The deficiency led to the use of empirical RBE factors in the clinical applications of particle therapy. The use of ionization dosimetry is similarly challenged when applied to development of radiation treatment at ultrahigh (FLASH) dose rate. This study reports a molecular method using plasmid DNA as a more comprehensive model for radiation dosimetry than ionization measurements. Aqueous solutions of purified supercoiled plasmid DNA, pUC19 (2686 bp), were prepared at different scavenging conditions and injected into 5x5x1 mm3 wells as detector elements. Irradiated samples were analyzed using base excision repair enzymes (Nth and Fpg) and gel electrophoresis to measure yields for DNA single and double strand breaks (SSB and DSB), and clustered lesions. The low LET characteristics of conventional radiation treatment was modeled using orthovoltage 150 kVp x-rays to deliver 2-110 Gy at 90 and 0.5 Gy/s. Higher LET irradiations in the range of 2 - 14 keV/μm were facilitated by measurements in the pristine Bragg peak region using synchrotron-produced 142.2 MeV protons to deliver dose at 2 - 160 Gy at 600 and 1 Gy/s. The DNA wells were inserted into a solid water equivalent phantom for proton irradiation. Only 4 wells could be positioned in the short Bragg peak region in water (∼ 2 cm). To alleviate the uncertainties due to rapidly varying dose and LET distributions, we innovated the use of a 3% water density (i.e., Styrofoam) medium to extend the Bragg peak region from 2 cm to 20 cm, enabling the placement of 20 well containers. Quantity and quality of molecular damage in the plasmid DNA model varies with fluence, fluence rate and LET of radiation. At high fluence (> 30 Gy) of low-LET radiations, the yields of DNA SSB and non-DSB clustered lesions depend on the fluence rate. These yields decrease by two times between ultrahigh and conventional dose rate irradiation. At a given fluence and fluence rate, the yields for the formation of DNA DSB and non-DSB clustered lesions increase linearly with LET. The low-density phantom allows significant (∼ 10 folds) increase in the number of sampling points and more accurate sample positioning at specific LET compared to water-equivalent phantom. Monte-Carlo track structure simulation of yields for different DNA lesions is being developed to model the molecular damage. In parallel, approaches to improve the sensitivity of the measurements to dose are being investigated. Our results suggest that a molecular-based approach can be used to differentiate the effects of fluence, fluence rate, and LET on radiation damage. The approach demonstrates the potential to improve on the modeling of radiation effects in biological systems than using measured ionization energy. Correlation of the molecular changes to biological outcome for in vitro and in vivo systems are under investigation.

Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγstrand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.

The Molecular Mechanisms of Actions, Effects, and Clinical Implications of PARP Inhibitors in Epithelial Ovarian Cancers: A Systematic Review

Ovarian cancer is the most lethal gynecologic malignancy in the United States. Some patients affected by ovarian cancers often present genome instability with one or more of the defects in DNA repair pathways, particularly in homologous recombination (HR), which is strictly linked to mutations in breast cancer susceptibility gene 1 (BRCA 1) or breast cancer susceptibility gene 2 (BRCA 2). The treatment of ovarian cancer remains a challenge, and the majority of patients with advanced-stage ovarian cancers experience relapse and require additional treatment despite initial therapy, including optimal cytoreductive surgery (CRS) and platinum-based chemotherapy. Targeted therapy at DNA repair genes has become a unique strategy to combat homologous recombination-deficient (HRD) cancers in recent years. Poly (ADP-ribose) polymerase (PARP), a family of proteins, plays an important role in DNA damage repair, genome stability, and apoptosis of cancer cells, especially in HRD cancers. PARP inhibitors (PARPi) have been reported to be highly effective and low-toxicity drugs that will tremendously benefit patients with HRD (i.e., BRCA 1/2 mutated) epithelial ovarian cancer (EOC) by blocking the DNA repair pathways and inducing apoptosis of cancer cells. PARP inhibitors compete with NAD+ at the catalytic domain (CAT) of PARP to block PARP catalytic activity and the formation of PAR polymers. These effects compromise the cellular ability to overcome DNA SSB damage. The process of HR, an essential error-free pathway to repair DNA DSBs during cell replication, will be blocked in the condition of BRCA 1/2 mutations. The PARP-associated HR pathway can also be partially interrupted by using PARP inhibitors. Grossly, PARP inhibitors have demonstrated some therapeutic benefits in many randomized phase II and III trials when combined with the standard CRS for advanced EOCs. However, similar to other chemotherapy agents, PARP inhibitors have different clinical indications and toxicity profiles and also face drug resistance, which has become a major challenge. In high-grade epithelial ovarian cancers, the cancer cells under hypoxia- or drug-induced stress have the capacity to become polyploidy giant cancer cells (PGCCs), which can survive the attack of chemotherapeutic agents and start endoreplication. These stem-like, self-renewing PGCCs generate mutations to alter the expression/function of kinases, p53, and stem cell markers, and diploid daughter cells can exhibit drug resistance and facilitate tumor growth and metastasis. In this review, we discuss the underlying molecular mechanisms of PARP inhibitors and the results from the clinical studies that investigated the effects of the FDA-approved PARP inhibitors olaparib, rucaparib, and niraparib. We also review the current research progress on PARP inhibitors, their safety, and their combined usage with antiangiogenic agents. Nevertheless, many unknown aspects of PARP inhibitors, including detailed mechanisms of actions, along with the effectiveness and safety of the treatment of EOCs, warrant further investigation.

Sperm DNA damage compromises embryo development, but not oocyte fertilisation in pigs

BackgroundThe assessment of sperm DNA integrity has been proposed as a complementary test to conventional mammalian semen analysis. In this sense, single-strand (SSB) and double-strand (DSB) DNA breaks, the two types of sperm DNA fragmentation (SDF), have been reported to have different aetiologies and to be associated to different fertility outcomes in bovine and humans. Considering that no studies in porcine have addressed how SDF may affect sperm quality and fertility outcomes, the present work aimed to determine the impact of global DNA damage, SSB and DSB on sperm quality and in vitro fertilising ability. To this end, 24 ejaculates (one per boar) were split into three aliquots: the first was used to assess sperm quality parameters through a computer-assisted sperm analysis (CASA) system and flow cytometry; the second was used to perform in vitro fertilisation, and the third, to evaluate sperm DNA integrity using alkaline and neutral Comet assays.ResultsThe results showed that global DNA damage negatively correlates (P < 0.05) with normal sperm morphology (R = − 0.460) and progressive motility (R = − 0.419), and positively with the percentage of non-viable sperm (R = 0.507). Multiple regression analyses showed that non-viable sperm were related to SSB (β = − 0.754). In addition, while fertilisation did not seem to be affected by sperm DNA integrity, global DNA damage, DSB and SSB were found to be correlated to embryo development outcomes. Specifically, whereas global DNA damage and DSB negatively affected (P < 0.05) the later preimplantation embryo stages (percentage of early blastocyst/blastocyst D6: for global DNA damage, R = − 0.458, and for DSB, R = − 0.551; and percentage of hatching/hatched blastocyst D6: for global DNA damage, R = − 0.505, and for DSB, R = − 0.447), global DNA damage and SSB had a negative impact (P < 0.05) on the developmental competency of fertilised embryos (R = − 0.532 and R = − 0.515, respectively). Remarkably, multiple regression analyses supported the associations found in correlation analyses. Finally, the present work also found that the inclusion of Comet assays to the conventional sperm quality tests improves the prediction of blastocyst formation (AUC = 0.9021, P < 0.05), but not fertilisation rates (P > 0.05).ConclusionConsidering all these findings, this work sets a useful model to study how SDF negatively influences fertility.

An in vivo Interaction Network of DNA-Repair Proteins: A Snapshot at Double Strand Break Repair in Deinococcus radiodurans.

An extremophile Deinococcus radiodurans survives massive DNA damage by efficiently mending hundreds of double strand breaks through homology-dependent DNA repair pathways. Although DNA repair proteins that contribute to its impressive DNA repair capacity are fairly known, interactions among them or with proteins related to other relevant pathways remain unexplored. Here, we report in vivo cross-linking of the interactomes of key DNA repair proteins DdrA, DdrB, RecA, and Ssb (baits) in D. radiodurans cells recovering from gamma irradiation. The protein-protein interactions were systematically investigated through co-immunoprecipitation experiments coupled to mass spectrometry. From a total of 399 proteins co-eluted with the baits, we recovered interactions among diverse biological pathways such as DNA repair, transcription, translation, chromosome partitioning, cell division, antioxidation, protein folding/turnover, metabolism, cell wall architecture, membrane transporters, and uncharacterized proteins. Among these, about 80 proteins were relevant to the DNA damage resistance of the organism based on integration of data on inducible expression following DNA damage, radiation sensitive phenotype of deletion mutant, etc. Further, we cloned ORFs of 23 interactors in heterologous E. coli and expressed corresponding proteins with N-terminal His-tag, which were used for pull-down assays. A total of 95 interactions were assayed, in which we confirmed 25 previously unknown binary interactions between the proteins associated with radiation resistance, and 2 known interactions between DdrB and Ssb or DR_1245. Among these, five interactions were positive even under non-stress conditions. The confirmed interactions cover a wide range of biological processes such as DNA repair, negative regulation of cell division, chromosome partitioning, membrane anchorage, etc., and their functional relevance is discussed from the perspective of DNA repair. Overall, the study substantially advances our understanding on the cross-talk between different homology-dependent DNA repair pathways and other relevant biological processes that essentially contribute to the extraordinary DNA damage repair capability of D. radiodurans. The data sets generated and analyzed in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD021822.

Strand Displacement and Unwinding Assays to Study the Concerted Action of the DNA Polymerase and SSB During Phi29 TP-DNA Replication.

The Bacillus subtilis phage Phi29 has a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA). Phi29 single-stranded DNA-binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated during the Phi29 genome replication, stimulating the DNA elongation rate. Here, we describe some protocols to evaluate the effect of Phi29 SSB mutants on the DNA elongation rate and their unwinding activity during replication by Phi29 DNA polymerase using as substrate TP-DNA and also singly primed M13 DNA.

Photo(geno)toxicity changes associated with hydroxylation of the aromatic chromophores during diclofenac metabolism

Diclofenac (DCF) can cause adverse reactions such as gastrointestinal, renal and cardiovascular disorders; therefore, topical administration may be an attractive alternative to the management of local pain in order to avoid these side effects. However, previous studies have shown that DCF, in combination with sunlight, displays capability to induce photosensitivity disorders. In humans, DCF is biotransformed into hydroxylated metabolites at positions 4′ and 5 (4′OH-DCF and 5OH-DCF), and this chemical change produces non negligible alterations of the drug chromophore, resulting in a significant modification of its light-absorbing properties. In the present work, 5OH-DCF exhibited higher photo(geno)toxic potential than the parent drug, as shown by several in vitro assays (3T3 NRU phototoxicity, DNA ssb gel electrophoresis and COMET), whereas 4′OH-DCF did not display significant photo(geno)toxicity. This could be associated, at least partially with their more efficient UV-light absorption by 5OH-DCF metabolite and with a higher photoreactivity. Interestingly, most of the cellular DNA damage photosensitized by DCF and 5OH-DCF was repaired by the cells after several hours, although this effect was not complete in the case of 5OH-DCF.

DNA binding is essential for PprI function in response to radiation damage in Deinococcus radiodurans

The extremely radioresistant bacterium Deinococcus radiodurans possesses a rapid and efficient but poorly known DNA damage response mechanism that mobilizes one-third of its genome to survive lethal radiation damage. Deinococcal PprI serves as a general switch to regulate the expression of dozens of proteins from different pathways after radiation, including the DNA repair proteins RecA, PprA and SSB. However, the underlying mechanism is poorly understood. In this study, we analyzed the dynamic alteration in global transcriptional profiles in wildtype and pprI mutant strains by combining microarrays and time-course sampling. We found that PprI up-regulated transcription of at least 210 genes after radiation, including 21 DNA repair and replication-related genes. We purified PprI and a helix-turn-helix (HTH) domain mutant and found that PprI specifically bound to the promoters of recA and pprA in vitro but did not bind nonspecific double-strand DNA. Chromatin immunoprecipitation (ChIP) assays confirmed that PprI specifically interacted with the promoter DNA of recA and pprA after radiation. Finally, we showed that a DNA-binding activity-deficient pprI mutant only partially restored resistance of the pprI mutant strain to γ radiation, UV radiation, and mitomycin C. Taken together, these results indicate that DNA-binding activity is essential for PprI to program the DNA repair process and cellular survival of D. radiodurans in response to radiation damage.

PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.

Low-dose hyper-radiosensitivity of progressive and regressive cells isolated from a rat colon tumour: Impact of DNA repair

Purpose: To ask whether highly metastatic sublines show more marked low-dose hyper-radiosensitivity (HRS) response than poorly metastatic ones.Materials and methods: The progressive (PRO) subline showing tumourigenicity and metastatic potential and the regressive (REG) subline showing neither tumourigenicity nor metastatic potential were both isolated from a parental rat colon tumour. Clonogenic survival, micronuclei and apoptosis, cell cycle distribution, DNA single- (SSB) and double-strand breaks (DSB) induction and repair were examined.Results: HRS phenomenon was demonstrated in PRO subline. Before irradiation, PRO cells show more spontaneous damage than REG cells. After 0.1 Gy, PRO cells displayed: (i) More DNA SSB 15 min post-irradiation, (ii) more unrepaired DNA DSB processed by the non-homologous end-joining (NHEJ) and by the RAD51-dependent recombination pathways, (iii) more micronuclei, than REG cells while neither apoptosis nor p53 phosphorylation nor cell cycle arrest was observed in both sublines.Conclusions: HRS response of PRO subline may be induced by impairments in NHEJ repair that targets G1 cells and RAD51-dependent repair that targets S-G2/M cells. The cellular consequences of such impairments are a failure to arrest in cell cycle, the propagation of damage through cell cycle, mitotic death but not p53-dependent apoptosis. Tumourigenic cells with high metastatic potential may preferentially show HRS response.

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single‐stranded DNA binding protein with increased processivity

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

The increased amount of vitamin B12 in serum is needed to minimize the uracil misincorporation into DNA during folate supplementation

The aim of the study was to find out the effect of dietary intake of folate and folic acid supplementation on the level of uracil misincorporation into DNA. The DNA damage was evaluated in young women's leukocytes with uracil glycosilase modified comet assay. Folate intake was evaluated on the basis of food diaries recorded in 2 months. The folate and B12 concentrations were evaluated in serum. Most of the studied individuals had diet deficient in folate. Folic acid supplementation (400 μg/day for 4 weeks) brought the folate intake much above the RDI level and the serum folate level in each individual was much above the deficiency range. The positive correlation ( p =0, 00156) was found between DNA SSB and the amount of B12 in serum after folic acid supplementation. The uracil misincorporation was not correlated with folate level but there was a significant negative correlation ( p =0, 05) with serum vitamin B12 concentration. The relationship occurred only after folate supplementation. This can suggest that high dietary folate intake is not sufficient to protect the leukocytes against uracil misincorporation if serum B12 concentration is not adequate.

Effects of Chlorophyll-Related Compounds on Hydrogen Peroxide Induced DNA Damage within Human Lymphocytes

Chlorophylls (Chl's) are the most abundant natural plant pigments. Four chlorophyll-related compounds (CRCs), including chlorophyllide a and b (Chlide a and b) and pheophorbide a and b (Pho a and b), were investigated for their antioxidative capacities to protect human lymphocyte DNA from hydrogen peroxide (H(2)O(2)) induced strand breaks and oxidative damage ex vivo. Lymphocytes exposed to H(2)O(2) at concentrations of 10 and 50 microM revealed an increased frequency of DNA single-strand breaks (ssb's; as measured by the comet assay) and also an increased level of oxidized nucleoside (as measured by 8-hydroxydeoxyguanosine, 8-OHdG). All Chl's reduced the level of DNA ssb's and 8-OHdG within human lymphocytes following exposure to 10 microM H(2)O(2). Only Pho a and b were able to decrease DNA ssb's and 8-OHdG following treatment of lymphocytes with 50 microM H(2)O(2), in a concentration-dependent fashion. It was demonstrated herein that Pho a and b were more antioxidative than others. We applied DPPH free-radical scavenge assays in vitro, and got similar results. Pho a and b had higher ability in scavenging capacities than others. We conclude that water-extract Chl's are able to enhance the ability of human lymphocytes to resist H(2)O(2)-induced oxidative damage, especially for Pho a and b.

Comparison between X-ray Photon and Secondary Electron Damage to DNA in Vacuum

Both monolayer and thick (20 microm) films of dry pGEM-3Zf(-) plasmid DNA deposited on tantalum foil were exposed to Al Kalpha X-rays (1.5 keV) for various times in an ultrahigh vacuum chamber. For monolayer DNA, the damage was induced mainly by low energy secondary electrons (SEs) emitted from the tantalum. For the thick films, DNA damage was induced chiefly by X-ray photons. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis. The exposure curves for the formation of nicked circular (single strand break, SSB), linear (double strand break, DSB), and interduplex cross-link forms 1 and 2 were obtained for both monolayer and thick films of DNA, respectively. The lower limits of G values for SSB and DSB induced by SEs were derived to be 86 +/- 2 and 8 +/- 2 nmol J(-1), respectively. These values are 1.5 and 1.6 times larger than those obtained with 1.5 keV photons. The projected X-ray energy dependence of the low energy electron (LEE) enhancement factor for the SSB and DSB in monolayer DNA is also discussed. This new method of investigation of the SE-induced damage to large biomolecules allows direct comparison of the yield of products induced by high energy photons and LEEs under identical experimental conditions.

Cellular Response to Pulsed Low-dose Rate Irradiation in X-ray Sensitive Hamster Mutant Cell Lines

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.

Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus

Bivalve molluscs, particularly mussels, are sensitive biomarkers of aquatic ecosystem pollution. The tannins, water-soluble plant polyphenols, may play an important role in this environment and, mainly as a consequence of interaction with pollutants, their toxicity may change. We studied three naturally occurring compounds, tannic acid, ellagic acid and gallic acid, for their ability to modulate DNA damage produced by these tannins alone and in the presence of the oxidative stress inducer H 2O 2, in cells of the digestive gland of mussels ( Unio tumidus). After the treatment of the cells with polyphenols at different concentrations (1, 5, 15, 30, 60, 80, 100, 120, 180, 240 μM) and with hydrogen peroxide in the range of 0.04 and 0.1 mM, single-strand breaks (ssb) in DNA were investigated, using the comet assay. The ability of phenolic acids to decrease DNA damage through their antioxidant properties was also assessed. The results show that the phenols, which are known as antioxidative agents, could also act as pro-oxidants. They induced ssb in DNA of the digestive gland at concentrations higher that 10 μM, but lower doses (1 and 5 μM) did not contribute to the DNA damage. This study was also designed to evaluate the protective effect of these tannins against H 2O 2-mediated DNA damage in the cells. In this treatment, the two concentrations (1 and 5 μM) significantly decreased the amount of lesions induced by H 2O 2 (0.04 and 0.1 mM). In conclusion, our results demonstrate that antioxidative properties of tannins may change to pro-oxidative activities at the higher concentrations. This suggests that the biologic actions of these compounds may be rather complicated.

Biochemical Characterization of Interactions between DNA Polymerase and Single-stranded DNA-binding Protein in Bacteriophage RB69

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.

Homologous-pairing Activity of the Bacillus subtilisBacteriophage SPP1 Replication Protein G35P

Genetic evidence suggests that the SPP1-encoded gene 35 product (G35P) is essential for phage DNA replication. Purified G35P binds single-strand DNA (ssDNA) and double-strand (dsDNA) and specifically interacts with SPP1-encoded replicative DNA helicase G40P and SSB protein G36P. G35P promotes joint molecule formation between a circular ssDNA and a homologous linear dsDNA with an ssDNA tail. Joint molecule formation requires a metal ion but is independent of a nucleotide cofactor. Joint molecules formed during these reactions contain a displaced linear ssDNA strand. Electron microscopic analysis shows that G35P forms a multimeric ring structure in ssDNA tails of dsDNA molecules and left-handed filaments on ssDNA. G35P promotes strand annealing at the AT-rich region of SPP1 oriL on a supercoiled template. These results altogether are consistent with the hypothesis that the homologous pairing catalyzed by G35P is an integral part of SPP1 DNA replication. The loading of G40P at a d-loop (ori DNA or at any stalled replication fork) by G35P could lead to replication fork reactivation.